Project Summary The plasma membrane (PM) forms the physical barrier and functional interface between a cell and its environment. To accommodate this complexity, the functionality of the PM is amplified by compartmentalization into compositionally and functionally distinct lateral domains, of which lipid rafts are the archetypal example. Raft-mediated signal transduction has been extensively implicated in diverse cell functions, with dysregulation contributing to the aberrant signaling in cancer, hyperinflammation, autoimmunity, and cardiovascular disease. Despite this potential impact, a dearth of consistent, quantitative methodologies has prevented clear definition of raft composition or unequivocal mechanistic description of raft function. A recent methodological breakthrough is the direct observation of large-scale ordered domains in plasma membranes isolated from mammalian cells. This system confirms the inherent capacity of mammalian PMs to form raft domains and also provides a robust experimental platform for direct, quantitative investigations into their composition and physical properties. We propose a comprehensive approach combining biophysics, bioinformatics, in silico molecular modeling, and cell biology to characterize the structural determinants and functional consequences of protein partitioning to PM microdomains. Our extensive preliminary data reveal that protein transmembrane domains (TMDs) encompass the necessary determinants for raft affinity. In Aim 1, we will define the general TMD physical features that impart raft affinity, focusing specifically on TMD length and surface area to test the hypothesis that relatively long and thin TMDs have more favorable interactions with ordered membrane microenvironments. Experimental measurements of raft partitioning will be supported by computational modeling and bioinformatics with the ultimate goal of generating a physical model that can identify raft preferring proteins from amino acid sequence. In Aim 2, we will extend the study from single TMDs to evaluate the role of TMD oligomerization in driving raft affinity. Our preliminary data has identified a specific TMD sequence motif that significantly enhances raft phase association. We will evaluate the hypothesis that such enhancement is driven by TMD oligomerization via quantitative evaluation of TMD oligomerization and its effect on raft partitioning in live cells, isolated PMs, and synthetic model systems. The structural details behind these observations will be investigated by atomistic molecular modeling. Finally, we aim to definitively demonstrate raft affinity as a major regulator of subcellular membrane traffic by the experiments proposed in Aim 3. To this end, we have generated a panel of protein variants lacking any sorting determinants except their TMD-encoded raft affinity. For these proteins, PM recycling after endocytosis relies on their partitioning into ordered membrane domains, implying a raft- mediated protein sorting mechanism. The trafficking pathways and molecular machinery underlying this mechanism will be investigated by imaging experiments using the TMD panel as validated probes of raft and non-raft domains. These studies will identify proteins that rely on microdomain association for their function, define the physicochemical nature of this association, and clarify the mechanisms by which PM organization regulates cell physiology. The long-term goal is to facilitate rational design of small molecules that interfere with protein association with microdomains in disease states defined by aberrant PM signal transduction.